Metabolic Engineering of Saccharomyces cerevisiae for Betulinic Acid Biosynthesis Reveals Pitfalls of Mitochondrial Compartmentalization of the Mevalonate Pathway

Betulinic acid (BA) is a high-value post-squalene triterpenoid with various pharmacological activities, and microbial biosynthesis offers an alternative route to its production. Mitochondrial compartmentalization of the mevalonate (MVA) pathway has been widely adopted to boost squalene supply, yet its impact on post-squalene triterpenoid biosynthesis remains poorly characterized. Here, we systematically engineered Saccharomyces cerevisiae for efficient BA production via combinatorial strategies including enzyme screening, protein-peptide interaction-mediated multienzyme assembly, mitochondrial MVA pathway engineering and optimization of the cytochrome P450 (CYP450)/cytochrome P450 reductase (CPR) module. We first identified the RoCYP01 (CYP716A155)/RoCPR pair from Rosmarinus officinalis as a better catalyst for sequential C28 oxidation, outperforming the BPLO (CYP716A180)/LjCPR pair. A protein-peptide-based assembly strategy involving Olea europaea lupeol synthase (OeLUP) and RoCYP01 further increased BA titer by 27%. Strikingly, mitochondrial reconstruction of the MVA pathway substantially increased squalene accumulation but caused a sharp decline in post-squalene triterpenoids. Transcriptomic profiling identified changes in the expression of genes associated with protein folding and auxiliary electron transfer, suggesting possible perturbations of endoplasmic reticulum-associated processes. Relieving 2,3-oxidosqualene constraints and engineering reduced nicotinamide adenine dinucleotide phosphate (NADPH) metabolism failed to restore BA biosynthesis effectively. After removal of the mitochondrial MVA module and optimization of BA synthesis module copy numbers, the engineered strain C7P produced 102.26 ± 8.99 mg/L betulinic acid in shake flasks. Its auxotrophic marker complemented derivative C8P achieved 323 mg/L betulinic acid and 946 mg/L betulin in a 5 L fed-batch bioreactor. Collectively, this study establishes an engineered yeast strain with improved BA production and shows that the mitochondria-targeted MVA engineering did not improve BA production, despite increasing squalene accumulation. These findings highlight the need to evaluate mitochondria-targeted precursor engineering.